Super/subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases

The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31 degrees C and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.     

Separation of chiral sulfoxides by liquid chromatography using macrocyclic glycopeptide chiral stationary phases

A set of 42 chiral compounds containing stereogenic sulfur was prepared. There were 31 chiral sulfoxide compounds, three tosylated sulfilimines and eight sulfinate esters. The separations were done using five different macrocyclic glycopeptide chiral stationary phases (CSPs), namely ristocetin A, teicoplanin, teicoplanin aglycone (TAG), vancomycin and vancomycin aglycone (VAG) and seven eluents, three normal-phase mobile phases, two reversed phases and two polar organic mobile phases. Altogether the macrocyclic glycopeptide CSPs were able to separate the whole set of the 34 sulfoxide enantiomers and tosylated derivatives. Five of the eight sulfinate esters were also separated. The teicoplanin and TAG CSPs were the most effective CSPs able to resolve 35 and 33 of the 42 compounds. The three other CSPs each were able to resolve more than 27 compounds. The normal-phase mode was the most effective followed by the reversed-phase mode with methanol-water mobile phases. Few of these compounds could be separated in the polar organic mode with 100% methanol mobile phases. Acetonitrile was also not a good solvent for the resolution of enantiomers of sulfur-containing compounds, neither in the reversed-phase nor in the polar organic mode. The structure of the chiral molecules was compared to the enantioselectivity factors obtained with the teicoplanin and TAG CSP. It is shown that the polarity, volume and shape of the sulfoxide substituents influence the solute enantioselectivity factor. Changing the oxidation state of the sulfur atom from sulfoxides to sulfinate esters is detrimental to the compound's enantioselectivity. The enantiomeric retention order on the teicoplanin and TAG CSPs was very consistent: the (S)-(+)-sulfoxide enantiomer was always the less retained enantiomer. In contrast, the (R)-(-)-enantiomer was less retained by the ristocetin A, vancomycin and vancomycin aglycone columns, showing the complementarity of these CSPs. The macrocyclic glycopeptide CSPs provided broad selectivity and effective separations of chiral sulfoxides.     

"Click" preparation of hindered cyclodextrin chiral stationary phases and their efficient resolution in high performance liquid chromatography

This communication reports the preparation of two new cyclodextrin (CD) chiral stationary phases (CSPs): heptakis(6-deoxy-6-azido)-β-CD and heptakis(6-deoxy-6-azido-phenylcarbamoylated)-β-CD CSPs that perform quite differently to our previously reported “click” immobilized CD-CSPs. These CSPs are sterically congested at the narrow mouth of the CD and exhibit chiral discrimination between over 40 pairs of enantiomers in high performance liquid chromatography. The free hydroxyl CSP afforded better separation of indoprofen, ketoprofen, Tröger's base, hydroxyl, carboxylic and dansyl amino acids than did the phenylcarbamoylated CSP, while the latter was better at resolving aryl alcohols, flavonoids, β-blockers and β-agonists. The current work shows that enantiodiscrimination achieved with different CSPs for different classes of analyte may be correlated with CD accessibility and peripheral functionality.     

不同载体涂敷的纤维素酯手性固定相的制备及性能

以微晶纤维素和3,5-二甲基苯基氨基甲酸酯为原料,合成了纤维素-三(3,5-二甲基苯基氨基甲酸酯)(CDMPC),将其分别涂敷于小孔硅胶(SG)、氨丙基化小孔硅胶(APS-SG)和介孔SBA-15微球上,制得3种手性固定相：SG@CDMPC(CSP1)、APS-SG@CDMPC(CSP2)及SBA-15@CDMPC(CSP3),正相条件下考察了12种中性或酸性手性化合物在自制手性固定相上的拆分效果,并与商品柱Chiralcel OD-H的拆分性能进行了对比。 6个手性化合物在CSP1上获得比在商品柱上更高的柱效,其中2个手性化合物获得比在商品柱上更高的分离因子和分离度,而CSP2和CSP3的拆分效果总体较CSP1和商品柱的差。 探讨了自制手性固定相对华法令的拆分和定量测定,华法令在CSP1上的检测限为10 μg/L,在0.05~5 g/L范围内线性关系良好。     

Comparison of enantioseparation of selected benzodiazepine and phenothiazine derivatives on chiral stationary phases based on β‐cyclodextrin and macrocyclic antibiotics

Enantioselective separation of some phenothiazine and benzodiazepine derivatives was studied on six different chiral stationary phases (CSPs) in HPLC. Selected CSPs, with respect to the structure of the separated compounds, were either based on β‐cyclodextrin chiral selectors – underivatized β‐cyclodextrin and hydroxypropyl ether β‐cyclodextrin, or on macrocyclic antibiotics – vancomycin, teicoplanin, teicoplanin aglycone, and ristocetin A. Measurements were carried out in a reversed‐phase separation mode. The influence of mobile phase composition on retention and enantioseparation was studied. Benzodiazepines could be enantioresolved with almost all the chiral stationary phases used, except for the vancomycin‐bonded CSP. Peak coalescence of oxazepam and lorazepam was observed if separation was carried out at laboratory temperature. Reduced temperature was required in some instances in order to avoid the on‐column racemization. Separation systems composed of teicoplanin‐bonded CSP and buffer‐methanolic or pure methanolic mobile phases were shown to be suitable even for preparative purposes due to high resolution values of the enantiomers. Enantioseparation of phenothiazine derivatives was more difficult to achieve but it was successful, at least partly, also with both types of the CSPs used (except for levomepromazine).     

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R(S) = 0.81) were well resolved (R(S) > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26-40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation.     

Quick development of an analytical enantioselective high performance liquid chromatography separation and preparative scale-up for the flavonoid Naringenin

The HPLC enantioselective separation of (R/S)-Naringenin, a chiral flavonoid found in several fruits juices and well-known for its beneficial health-related properties, including antioxidant, anti-inflammatory, cancer chemopreventive, immunomodulating and antimicrobial activities, has been performed on both analytical and (semi)-preparative scale using an amylose derived Chiralpak AD chiral stationary phase (CSP). A standard screening protocol for cellulose and amylose based CSPs was firstly applied to analytical Chiralcel OD-H and Chiralpak AD-H, as well as to Lux Cellulose-1, Lux Cellulose-2 and Lux Amylose-2 in order to identify the best experimental condition for the subsequent scaling-up. Using Chiralpak AD-H and eluting with pure methanol (without acidic or basic additives) relatively short retention times, high enantioselectivity and good resolution (α=1.49, R(s)=3.48) were observed. Therefore, these experimental conditions were properly scaled-up to (semi)-preparative scale using both a pre-packed Regispack column and a Chiralpak AD column packed in house with bulk CSP. The developed preparative method proved to be superior to previously published methods in terms of elution times, separation and resolution and is suitable for obtaining a quick access to the desired enantiomers with high enantiomeric excess and amounts sufficient for biological investigations. Future scale-up options (enantioselective supercritical fluid chromatography or HPLC in the Simulated Moving Bed mode) were also evaluated. It could be shown that both methodologies have a high potential for future production of Naringenin enantiomers by enantioselective chromatography.     

Preparation and evaluation of new Pirkle type chiral stationary phases with long alkyl chains for the separation of amino acid enantiomers derivatized with NBD-F.

In order to improve the high-performance liquid chromatographic separation of alpha-amino acids derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) on commercially available chiral stationary phases (CSPs) such as SUMICHIRAL OA-2500(S) (CSP 1) and OA-4700 (CSP 3), the preparation of two new CSPs (CSP 2 and CSP 4) having 11-aminoundecanoic acid between the aminopropyl silica gel support and the chiral moiety in CSP 1 and CSP 3 is described. CSP 2 and CSP 4 improved both the mutual and enantiomeric separation of NBD-amino acids compared with CSP 1 and CSP 3. Thus, 17 pairs of NBD-amino acid enantiomers and NBD-glycine were separated on CSP 2 except for six NBD-amino acids (D-Asn, D-Ser, D-Gln, L-Pro, L-Ser and Gly). CSP 2 and CSP 4 also showed better enantiomeric separation of NBD-amino acid esters and amides than CSP 1 and CSP 3. It was considered that the achiral long alkyl chains in the CSPs might form a hydrophobic space which assisted the stereoselective interaction of analytes with the chiral moiety by changing the environment around the chiral moiety. On CSP 1 and CSP 2, NBD-beta-amino acid was also enantiomerically separated.     

Effect of the Orientation of Amide Linkage Groups on the Enantioselectivity of Two Related Synthetic Polymeric Chiral Stationary Phases

Two new synthetic polymeric chiral stationary phases (CSPs) based on trans-(1S,2S)-cyclohexanedicarboxylic acid bis-4-vinylphenylamide (I) and trans-N,N′-(1R,2R)-cyclohexanediyl-bis-4-ethenylbenzamide (II) monomers were prepared and evaluated by normal phase high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A variety of chiral compounds were separated on these two new CSPs. The different orientation of the amide groups in the two CSPs resulted in a striking difference in the enantioselectivity properties of these two CSPs. Their differences in enantioselectivity with HPLC and SFC were compared.     

Super/Subcritical Fluid Chromatography Separations with Four Synthetic Polymeric Chiral Stationary Phases

New synthetic polymeric chiral selectors were developed recently as chiral stationary phases. They were tested with supercritical fluid mobile phases made of CO₂ plus an alcohol modifier and 0.2% v/v trifluoroacetic acid. The polymeric N,N′-(1S,2S)-1,2-cyclohexanediyl-bis-2-propenamide (P-CAP), the polymeric N,N′-[(1R,2R)]-1,2-diphenyl-1,2-ethanediyl] bis-2-propenamide (P-CAP-DP), the polymeric trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide (DEABV) and the polymeric N,N′-[(1R,2R)-1,2-diphenyl-1,2-ethanediyl] bis-4-vinylbenzamide (DPEVB) were bonded to 5 μm silica particles and used to prepare four columns that were tested with a set of 88 chiral compounds with a wide variety of chemical functionalities. All 88 test compounds were separated on one or more of these “related” polymeric CSPs. Forty-three enantiomeric pairs were separated in SFC conditions by only one of the CSPs. Twenty pairs were separated by two CSPs and 18 and 7 enantiomeric pairs were separated by 3 and all 4 CSPs, respectively. The three P-CAP, P-CAP-DP and DEABV CSPs have equivalent success being able to separate 49 enantiomeric pairs of the studied set with respectively 12, 14 and 20 at baseline (R _s  > 1.5). The DPEVB CSP was significantly less efficient separating only 18 chiral compounds with only one at baseline. The great advantage of the SFC mobile phases is the rapid separation, witch most achieved in less than 5 min.     

卵类粘蛋白柱手性拆分酮基布洛芬

 在卵类粘蛋白柱上 ,考察了酮洛芬对映体在 3种流动相体系下的保留行为 ,建立了同时分离酮洛芬及其甲酯两对对映体的较佳色谱条件。方法可用于酶法立体选择性水解或酯化过程中生成的 4种化合物的同时测定     

Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters by capillary gas chromatography on derivatized cyclodextrin stationary phases

Summary Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters has been investigated on derivatized cyclodextrin chiral stationary phases (CD CSPs) to enable direct determination of the enantiomeric purity of the products of enantioselective cyclopropanation. Four stereoisomers of these chiral compounds could be resolved to baseline on permethylated β-cyclodextrin CSP. Some unusual phenomena, iso-enthalpy retention behavior and entropically driven chiral separation, were observed for the enantioseparation of 2-phenylcyclo-propanecarboxylates on the CD CSPs. Thermodynamic parameters were evaluated and an enthalpy-entropy compensation effect was observed forn-alkyl esters of 2-phenylcyclopropanecarboxylate separated on CD CSPs.     

Chromatographic Resolution of α‐Amino Acids by (R)‐(3,3'‐Halogen Substituted‐1,1'‐binaphthyl)‐20‐crown‐6 Stationary Phase in HPLC

Three new chiral stationary phases (CSPs ) for high‐performance liquid chromatography were prepared from R ‐(3,3'‐halogen substituted‐1,1'‐binaphthyl)‐20‐crown‐6 (halogen = Cl, Br and I). The experimental results showed that R ‐(3,3'‐dibromo‐1,1'‐binaphthyl)‐20‐crown‐6 ( CSP ‐1 ) possesses more prominent enantioselectivity than the two other halogen‐substituted crown ether derivatives. All twenty‐one α ‐amino acids have different degrees of separation on R ‐(3,3'‐dibromo‐1,1'‐binaphthyl)‐20‐crown‐6‐based CSP ‐1 at room temperature. The enantioselectivity of CSP ‐1 is also better than those of some commercial R ‐(1,1'‐binaphthyl)‐20‐crown‐6 derivatives. Both the separation factors (α ) and the resolution (R _s) are better than those of commercial crown ether‐based CSPs [CROWNPAK CR (+) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and valine, which cannot be separated by commercial CR (+). This study proves the commercial usefulness of the R ‐(3,3'‐dibromo‐1,1'‐binaphthyl)‐20‐crown‐6 chiral stationary phase.     

Capillary electrochromatographic separation of enantiomers on chemically bonded type of cellulose derivative chiral stationary phases with a positively charged spacer

Positively charged chiral stationary phases (CSPs) were prepared for capillary electrochromatography (CEC) separation of enantiomers by chemically immobilizing cellulose derivatives onto diethylenetriaminopropylated silica (DEAPS) with tolylene-2,4-diisocyanate (TDI) as a spacer reagent. Anodic electroosmotic mobility was observed in both nonaqueous and aqueous mobile phases due to the positively charged amines on the surface of the prepared CSPs. For comparison, the traditionally used 3-aminopropyl silica (APS) was also adopted as the base material instead of DEAPS to prepare CSP. It was observed that the EOF on the DEAPS-based CSP was 18%-60% higher than that on the APS-based CSP under nonaqueous mobile phase conditions. Separation of enantiomers in CEC was performed on the positively charged CSPs with the nonaqueous mobile phases of pure ethanol or mixture of hexane-alcohol and the aqueous phases of acetonitrile-water or 95% ethanol. Fast separation of enantiomers was achieved on the newly prepared CSPs.     

Evaluation of the macrocyclic glycopeptide A-40,926 as a high-performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.     

Synthesis and chromatographic evaluation of new polymeric chiral stationary phases based on three (1<Emphasis Type="Italic">S</Emphasis>,2<Emphasis Type="Italic">S</Emphasis>)-(−)-1,2-diphenylethylenediamine derivatives in HPLC and SFC

Three new polymeric chiral stationary phases were synthesized based on (1S,2S)-1,2-bis(2,4,6-trimethylphenyl)ethylenediamine, (1S,2S)-1,2-bis(2-chlorophenyl)ethylenediamine, and (1S,2S)-1,2-di-1-naphthylethylenediamine via a simple free-radical-initiated polymerization in solution. These monomers are structurally related to (1S,2S)-1,2-diphenylethylenediamine which is the chiral monomer used for the commercial P-CAP-DP polymeric chiral stationary phase (CSP). The performance of these three new chiral stationary phases were evaluated in normal phase high-performance liquid chromatography (HPLC) and supercritical fluid chromatography and the results were compared with those of the P-CAP-DP column. All three new phases showed enantioselectivity for a large number of racemates with a variety of functional groups, including amines, amides, alcohols, amino acids, esters, imines, thiols, and sulfoxides. In normal phase, 68 compounds were separated with 28 baseline separations (Rs ≥ 1.5) and in SFC, 65 compounds were separated with 24 baseline separations. In total 72 out of 100 racemates were separated by these CSPs with 37 baseline separations. Complimentary separation capabilities were observed for many analytes. The new polymeric CSPs showed similar or better enantioselectivities compared with the commercial column in both HPLC and SFC. However, faster separations were achieved on the new stationary phases. Also, it was shown that these polymeric stationary phases have good sample loading capacities while maintaining enantioselectivity.     

Correlation method for comparison of selectivity and retention on different chiral stationary phases

Summary The main chromatographic properties: selectivity and retention of two chiral stationary phases: (R)-3,5-dinitrobenzoylphenylglycine (CSP I) and (S)-3,5-dinitro-benzoylleucine (CSP II) have been compared on the basis of correlation of retention factors of derivatized esters of amino acids and derivatized aminoalcohols. The differences in retention and selectivity for the two CSPs can be easily estimated from the correlation equation. It is shown that in the case of the correlation for two sets of retention factors obtained on two different CSPs, it is mainly the intercept that decides which CSP has the better selectivity. It is demonstrated that the correlation method provides more informations on the selectivity and retention than comparison of the chromatographic data of single pairs of enantiomers. Additionally, the possible influence of the polar modifier mixed with n-hexane as diluent on the constants in the correlation equation is demonstrated.     

键合型聚丙烯酰胺衍生物手性固定相的制备与手性识别性能

高效液相色谱(HPLC)被广泛认为是分离制备光学纯单一对映体的最有效方法。在高效液相色谱手性拆分中,手性固定相(CSP)的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,已经发展成为一类重要的手性固定相。本文通过两步化学反应合成了新型的光学活性丙烯酰胺衍生物--(S)-1-丙烯酰-2-(N-苯基甲酰胺基)吡咯烷((S)-APACP),采用核磁共振氢谱表征了(S)-APACP的化学结构;通过3步化学反应制备了键合型聚丙烯酰胺衍生物手性固定相,采用热重分析法表征了聚合物的键合量,采用HPLC评价了键合型手性固定相的识别能力,分析了影响其手性识别能力的因素。研究结果表明,APACP聚合物成功地键合到硅胶表面制备了具有良好溶剂耐受性的键合型手性固定相,其聚合物键合量为10.2%~11.8%,该键合型手性固定相对若干种对映体显示了较好的手性识别能力。     

Applications of Cellulose-Based Chiral Stationary Phases in the Resolution of Some Beta-Adrenoceptor Antagonists

Abstract

Cellulose tris (3,5-dimethylphenyl carbamate) known as Chiralcel OD chiral stationary phase (CSP) is one of the most commonly used cellulosic CSPs which have been successfully used for separation, enantiomeric purity determination and analysis of several drug racemates including β-adrenoceptor antagonists.

Resolution of timolol, penbutolol, celiprolol and carazolol enantiomers are achieved using this CSP. A possible chiral recognition mechanism(s) for these β-adrenergic blockers and this chiral stationary phase is presented.